Polyphenols are natural products with important applications in the cosmetic, pharmaceutical and food industry (Quideau et al., 2011; Espín et al., 2007). In particular, resveratrol (3,5,4′-trihydroxystilbene) is seen as a very interesting compound, since several studies reported it to be anti-carcinogenic, anti-inflammatory, anti-microbial and even to expand man's life-span (Frémont, 2000; Novelle et al., 2015; Park and Pezzuto, 2015; Biagi and Bertelli, 2015). Glycosylation of resveratrol can dramatically improve its solubility and bioavailability, but the effect differs with the type of glycosidic bond (Thuan and Sohng, 2013; Torres et al., 2011; Ozaki et al., 2011). For example, the 3-α-glucoside of resveratrol was found to be more soluble than the 3-β-glucoside (Torres et al., 2011). For stereo- and regiospecific glycosylation, the advantages of biocatalysis over chemical processes are obvious (de Roode et al., 2003; Desmet et al., 2012; Bornscheuer et al., 2013).
Most glycosyl transferases require nucleotide-activated donor substrates (e.g. UDP-Glc), which are rather expensive for large-scale applications (Bungaruang et al., 2013; Breton et al., 2012; Gantt et al., 2011; Palcic, 2011; Lairson et al., 2008). However, a few enzymes are able to catalyze transglycosylation reactions starting from simple sugars like maltodextrins (e.g. CGTase) or sucrose (e.g. glucansucrase) (Desmet et al., 2012). Similarly, sucrose phosphorylase (SP, EC 2.4.1.7) can transfer the glucose moiety of sucrose to a variety of acceptors through a double displacement mechanism (Puchart, 2015; Desmet and Soetaert, 2012; Goedl et al., 2008; Aerts, Verhaeghe, Roman, Stevens, Desmet and Soetaert, 2011a). It should be noted that sucrose not only is cheap but also very reactive, with an energy content that is comparable with that of nucleotide sugars (Monsan et al., 2010). High yields can thus be obtained, as has been reported for the production of 2-O-alpha-D-glucopyranosyl-sn-glycerol, a moisturizing agent in cosmetics commercialized under the tradename Glycoin® (Goedl et al., 2008).
Unfortunately, the activity of SP on (poly)phenolic acceptors is very low, even barely detectable in most cases (Aerts, Verhaeghe, Roman, Stevens, Desmet and Soetaert, 2011b; Kitao et al., 2014; De Winter et al., 2014; De Winter et al., 2013). It has been suggested that the main problem is the enzyme's low affinity for such compounds, which cannot be compensated by increasing the acceptor concentrations because of their low solubility (De Winter et al., 2013; Aerts, Verhaeghe, Roman, Stevens, Desmet and Soetaert, 2011a). Although increased transglycosylation rates have been reported upon addition of organic solvents (De Winter et al., 2014; De Winter et al., 2013). However, saturation of the enzyme has never been achieved and productivity has generally remained too low for practical applications. With resveratrol, for example, the best result has been obtained in 20% of the ionic liquid AMMOENG™ 101, but that only generated 3 mM of resveratrol 3-alpha-glucoside (De Winter et al., 2013). Therefore, there is an urgent need to find or design an SP capable of efficiently glycosylating polyphenols such as resveratrol.
Two (thermo)stable SP enzymes have so far been identified, i.e. one in Bifidobacterium adolescentis (BaSP, Cerdobbel et al., 2011; WO2011/124538) and one in Thermoanaerobacterium thermosaccharolyticum (TtSPP, WO2014/060452). The latter enzyme actually prefers sucrose 6′-phosphate as substrate (EC 2.4.1.329) but can still efficiently use sucrose as glycosyl donor (Km=77 mM) (Verhaeghe et al., 2014). The latter authors further disclosed a R134A variant of TtSPP, which was shown to play a role in the binding of sucrose-6′-phosphate or fructose-6-phosphate.